Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Mycobacterium tuberculosis (M.tb), the causative agent of TB, is capable of establishing primary infections that often become latent and can persist despite an intact immune system. It is well known that M.tb possesses multiple highly evolved mechanisms to manipulate host cellular machinery and evade the host immune system. We have contributed to expanding the knowledge of M.tb physiology through the discovery of an alternative antigen release mechanism based on membrane vesicles (MV). Compositional analysis of MV revealed enrichment in polar lipids and lipoproteins. MV release events were visualized during in vitro and in vivo infections. MV stimulation of macrophages and mouse elicited inflammatory response dependent on Toll-like receptor 2 (TLR-2). The combination of MV stimulation with an aerosolized infection in mice produced a `Koch phenomenon' promoting the growth of mycobacteria in lungs and spleen, suggesting a link between MV and M.tb pathogenesis. Nevertheless a stronger association between vesiculation and virulence in M.tb remains to be established. We recently contributed to characterizing the phenotype of the Rv0431 M.tb mutant, which manifested a hyper- inflammatory response in macrophages due to overproduction of MV. This finding suggests that MV production is a regulated process and given the intrinsic complexity of the process, additional genes are likely to be involved. This application proposes a molecular dissection of vesiculogenesis in M.tb. Initially, we will identify genes responsible for vesicle production by screening a library of M.tb transposon insertion mutants using a sensitive, high-throughput dot blot system to detect MV in low-volume cultures. The screening assay uses antiserum capable of recognizing vesicles whose specificity is imparted by the physical separation techniques used to prepare the immunogen. Mutants with increased MV production, low MV production and non-producing mutants will be characterized by microscopic methods to gain insight into ultrastructural changes, and biochemical and mass spectrometry based-techniques will be employed to identify potentially altered MV composition. Further, we will investigate the role of Rv0431 in vesiculogenesis. The approach proposed here will allow identification of additional genetic variants affected in vesicle production, which willbe essential for studying their role in the pathogenesis of mycobacterial infection.